Next generation of AAV vector targeting adipose tissue

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. Adeno-associated viral (AAV) vectors are mainstream delivery platforms in gene therapy because of its safety profile and promising results in clinical trials. AAV has been successfully used to deliver transgenes to liver, heart, skeletal muscle, brain, and eye. Yet AAV-mediated gene transfer to adipose tissue is left far behind due to the low transduction efficiency and tropism of natural AAV serotypes. Our previous study demonstrates that an engineered hybrid capsid Rec2, transduces adipose tissue with superior efficiency than natural AAV serotypes. One caveat is that Rec2 capsid vector also transduces liver efficiently upon systemic administration. To mitigate off-target transgene expression in the liver, we have developed a novel dual-cassette vector design to severely restrict transgene expression in the liver. Although the dual-cassette design coupled with Rec2 capsid significantly improve the efficiency and selectivity of AAV-mediated gene delivery to adipose tissue, there are drawbacks such as the presence of Rec2 vectors in the liver and therefore bearing the risk of viral vector-associated toxicity; inability to suppress transgene expression in the liver if miRNA and shRNA are the transgene; the dual-cassette further reducing the cargo size. The weaknesses of existing technology highlight the need to generate new capsids with enhanced adipose tropism while eliminating liver tropism. Recently, we made several mutations in the Rec2 capsid to alter the tropism. Eight capsid variants (named LC.V1~8) have been generated, packaged, and tested in vivo. The capsid variant LC.V7, with three point-mutations, was found to possess exclusive adipo-tropism as it achieved substantial transduction in adipose tissue while completely eliminating liver transduction. Moreover, LC.V7 capsid vector did not transduce heart, spleen, muscle, kidney, or pancreas. Dosing experiment found that 4 x 1010 viral genome (vg) per mouse via intraperitoneal injection was sufficient for LC.V7 to transduce visceral fat and no transgene expression was detected in liver or any other peripheral tissues. Of note, 4 x 1010 vg/mouse is a low dose relative to systemic use of natural AAV serotypes often in the range of 1011 to 1012. In this project, we propose to develop the next generation of AAV vector platform for adipose gene delivery based on the novel LC.V7 capsid. In Aim 1, we will comprehensively characterize LC.V7 vector including biodistribution, administration routes, liver toxicity, and durability of transgene expression in lean and obese states. We will improve AAV genomic design to enhance transgene expression in adipose tissue. In Aim 2, we will develop LC.V7-based gene therapies for lipodystrophy. We will clone human leptin and human adiponectin cDNA sequence to the optimized AAV expression plasmid using a 2A sequence to express these two adipokines from one transcript and test the efficacy and safety of this gene therapy in two lipodystrophy models. This novel adipo-tropic AAV vector platform could provide a powerful tool for basic research and therapeutic purposes.